We propose to continue our studies on the mechanisms controlling the expression of the homeotic genes in the Bithorax Complex (BX-C). For the past decade we have been focusing on two types of cis-acting elements in BX-C hat are critical for the proper regulation of homeotic gene expression. The first are the Polycomb Response Elements PREs) while the second are the domain boundary elements. The studies proposed in this application are a continuation of this ongoing investigation and are divided into three parts. The studies in Part I are on the iab-7 PRE. During the previous grant period we defined the cis-acting sequences required for the silencing activity of the iab-7 PRE. We also used two different genetic screens to identify key trans-acting factors. During the upcoming grant period we plan to characterize several new Pc-G genes that we have shown in genetic experiments to be important for the silencing activity of this particular PRE. To complement this genetic analysis, we propose to extend our in vitro studies on the assembly of Pc-G complexes on a minimal iab-7 PRE element called HS3. We also proposed to study an unusual allele of Enhancer of Zeste that ectopically silences domains that should be active. This Pc-G protein appears to be inappropriately recruited to sites for trx/ashl located adjacent to the minimal iab-7 PRE. The experiments in Part II focus on the grappa protein, grappa was identified as a dominant suppressor of silencing mediated by the Mcp element and by the iab-7 PRE. The grappa gene is unusual in that in addition to being required for Pc-G mediated silencing, mutations in grappa also have trx-G like phenotypes. We have cloned grappa and found that it encodes a homolog of the yeast Dotl histone H3 K79 methylase. The major goal of the proposed studies is to understand why grappa has both Pc-G and trx-G like properties, and what function it plays in development and gene regulation. The experiments in Part III are on the Fab-7 boundary. The major goal of this project has been to identify and characterize proteins that are critical for Fab-7 boundary function, and analyze the genes that encode these proteins. In two independent genetic screens, we have identified a gene called pbro that is required for Fab-7 boundary function. During the upcoming grant period, we proposed to characterize the pbro gene. [unreadable] [unreadable]